All of the cells in the peripheral blood are generated from a small population of hematopoietic stem cells (HSC) through a process of proliferation and differentiation known as hematopoiesis. The hematopoietic differentiation program includes well-defined stages at which the progeny of HSC become restricted to specific fates. The goal of this project is to define specific molecular signatures associated with specific stages of hematopoiesis. Project 1. Epigenetic control of hematopoiesis - methylation. Epigenetic changes, including DNA methylation, are an essential element of normal development and hematopoietic differentiation. Our goal is to comprehensively map these changes in the genomes of primary mouse erythroblasts, megakaryocytes and their common progenitor, the MEP. The ENCODE project has given us a roadmap of changes in the epigenetic code of different cell lines, but to move ENCODE further, we have decided to focus on primary mouse erythroblasts, megakaryocytes and MEP isolated from bone marrow. Following up on our initial analysis of genome-wide methylation (Hogart A, Lichtenberg J, Ajay SS, Anderson S, NIH Intramural Sequencing Center, Margulies EH, Bodine DM. Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites. Genome Res. 22 (8) 1407-18, 2012), we now have shown that while methylation decreases as erythroid cells mature, increased methylation is associated with differentiation in the megakaryocyte and granulocyte lineages. We have developed new software packages to analyze these data that are becoming widely used in the field. (Lichtenberg J, Hogart A, Battle S. Bodine DM. Discovery of motif-based regulatory signatures in whole genome methylation experiments. Bioinformatics Open Source Conference (BOSC), 2012, Long Beach USA). Project 2. Epigenetic control of hematopoiesis transcription factor occupancy. We have entered into a consortium with a group at Penn State led by Ross Hardison to take advantage our respective labs' strengths. Our group is generating methylation and transcription factor profiles for erythroblasts, megakaryocytes and MEP and we have provided the Hardison lab with cells and chromatin for RNA-Seq analysis of the trancriptome and histone methylation. We have completed RNA-Seq analyses of erythroblasts, megakaryocytes and MEP. Our findings include the identification of over 600 novel long non-coding RNAs, many of which are expressed specifically in one of the three cell types. We plan to extend these analyses by correlating the changes in gene expression we observe with the changes in the epigenetic profile of the genome that we are developing in a different project. This will allow for the first time direct comparisons of gene expression and epigenetic signatures in primary cells.